Abstract

Selective δ-opioid agonists produce delayed cardioprotection that lasts for 24–48 h in rats; however, the maximum length of the cardioprotective window is unclear. In this study, we attempted to prolong the cardioprotective window using a unique δ-opioid agonist, fentanyl isothiocyanate (FIT), which binds irreversibly to the δ-receptor, and determined the role of the phosphatidylinositol 3-kinase (PI3K) pathway as a trigger or end effector of FIT-induced cardioprotection. Initially, male rats were administered FIT (10 μg/kg) 10 min before hearts were subjected to 30 min of ischemia and 2 h of reperfusion followed by infarct size (IS) assessment. Acute FIT administration reduced IS when given before ischemia, 5 min before reperfusion, or 10 s after reperfusion compared with control. IS reduction also occurred following a single dose of FIT at 48, 72, 96, and 120 h after administration vs. control, with the maximum effect observed at 96 h. FIT-induced IS reduction at 96 h was completely abolished when the irreversible PI3K inhibitor wortmannin (15 μg/kg) was given before FIT during the trigger phase; however, the effect was only partially abrogated when wortmannin was given 96 h later. These data suggest that FIT has a prolonged cardioprotective window greater than that of any previously described cardioprotective agent that requires PI3K primarily in the trigger phase but also partially, as a mediator or end effector.

delayed cardioprotection

phosphatidylinositol 3-kinase

opioids

BW373U86

postconditioning

the window of protection produced by ischemic preconditioning (IP) occurs in two phases: acutely, lasting up to 3 h, and through a delayed window of protection, lasting from 24 to 72 h (2). The delayed window of cardioprotection also exists for many pharmacological agents, including adenosine, bradykinin, ATP-sensitive K+ (KATP) channel openers, and opioids, with these agents providing a window of protection that also persists for 24–72 h (3, 9, 10, 13, 31, 38). This phenomenon also extends to human atrial myocardium, with protection occurring for up to 72 h by IP or pharmacological agents (27). Therefore, it has been assumed that the duration of delayed cardioprotection, following a single ischemic or pharmacological intervention, lasts for only 72 h.

Exogenous administration of selective δ-opioid agonists produces a reduction of infarct size when given acutely, before ischemia or immediately before reperfusion, and these compounds also produce a delayed effect when given 24–48 h before index ischemia (10, 14, 15, 25, 31, 37). The longest known window of cardioprotection by opioids occurred after TAN-67 administration, which lasted for 48 h; however, the duration of opioid-induced cardioprotection has not been tested with other δ-opioid agonists (10).

Fentanyl is a potent μ-opioid receptor agonist that has been shown to reduce infarct size in rats via a δ-opioid-dependent mechanism; however, it failed to improve postischemic mechanical function from stunning (4, 21, 22). Addition of cyanide to fentanyl, generating the compound fentanyl isothiocyanate (FIT), creates a selective, irreversible δ-opioid agonist that alkylates the δ-receptor without cross-reactivity with the μ-receptor (7, 36) (Fig. 1). The cardioprotective properties of FIT have not been previously determined.

Molecular structure of fentanyl and the fentanyl derivative, fentanyl isothiocyanate (FIT). Addition of a cyanide to fentanyl generates an irreversible δ-opioid agonist.

Acute protection by opioids was recently found to occur via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway, with phosphorylation of glycogen synthase kinase-β (GSK-β) at Ser9, which causes GSK-β inactivation (15). However, it is unknown whether the PI3K pathway mediates the trigger or end-effector phases of delayed δ-opioid-induced infarct size reduction.

Therefore, we examined the dose, length of the cardioprotective window, and dependence of the PI3K pathway on FIT-induced infarct size reduction in rats. We further examined whether FIT induces the activation of the PI3K pathway.

METHODS

The experimental procedures and protocols used in this study were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin and conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Male Sprague-Dawley rats (215–300 g) were obtained from Harlan and used for an in vivo anesthetized, intact rat model of ischemia and reperfusion consisting of 30 min of ischemia and 2 h of reperfusion. The general surgical protocols for acute and delayed infarct size studies, including the determination of infarct size, have been described previously in detail (13). Hemodynamics, including heart rate, mean arterial pressure, and rate-pressure product, were quantified during baseline, 15 min into ischemia, and at 2 h of reperfusion.

Pharmacological agents.

The agents used for this study included FIT {N-1-[2-(4-isothiocyanatophenyl)ethyl]-4-piperidinyl-N-phenylpropanamide; Tocris}, BW373U86 (Ardent), and wortmannin (Sigma). FIT was dissolved in 95% ethanol, whereas BW373U86 and wortmannin were dissolved in DMSO.

Acute infarct size studies.

For the acute infarct size studies, FIT was administered 10 min before ischemia at a range of doses (1–300 μg/kg) and infarct size was determined. Additional rats received vehicle (95% ethanol) 10 min before ischemia. The optimal dose of FIT necessary to produce the maximal reduction of infarct size compared with the vehicle-treated group was determined in this series of experiments.

A second series of experiments determined whether FIT (10 μg/kg) administered 5 min before reperfusion or 10 s after reperfusion could reduce infarct size equal to that measured when FIT was administered 10 min before ischemia.

Delayed infarct size studies.

Rats were treated with a single intravenous dose of FIT (10 μg/kg) via tail vein injection. Animals were provided with ample food and water and allowed to recover for time periods ranging from 48 to 144 h. An additional series of rats was treated with a single intravenous dose of BW373U86 (10 μg/kg), another selective δ-opioid agonist, and allowed to recover for 96 h. Rats were then subjected to 30 min of ischemia and 2 h of reperfusion, followed by infarct size assessment.

A second series of rats was treated with the PI3K inhibitor wortmannin (15 μg/kg) 90 min before FIT (10 μg/kg) administration to determine whether triggering of FIT-induced infarct size reduction is dependent on the PI3K pathway. Rats were allowed to recover for 96 h and then subjected to ischemia-reperfusion and infarct size assessment. To determine the contribution of the PI3K pathway as a mediator or end effector of FIT-induced infarct size reduction, we administered FIT (10 μg/kg) and allowed rats to recover for 96 h. Wortmannin was administered 10 min before index ischemia, and the standard ischemia-reperfusion and infarct size protocols were conducted.

Statistical significance.

All values are denoted as means ± SE. For the hemodynamics and infarct size studies, statistical significance was determined by performing a one-way ANOVA with Bonferroni's correction for multiplicity. A significant difference was indicated at a P value <0.001 or <0.01.

RESULTS

We used 131 rats to perform 119 successful experiments. Twelve rats were omitted because of ventricular fibrillation during reperfusion (3), a suture breaking during negative staining for the normal zone (2), or failure to administer the full drug dose into the tail vein because of the rat flicking its tail (7).

Hemodynamics.

Heart rate, mean arterial pressure, and rate-pressure product for the acute infarct size experiments showed significant differences in baseline heart rate between the vehicle-treated group and the experimental group when FIT was administered 10 s after reperfusion. Significant differences in mean arterial pressure were also present between the vehicle-treated group and the experimental group when FIT was administered at 300 μg/kg before ischemia and when FIT was administered 5 min before reperfusion. No other significant differences were seen in hemodynamics for the acute infarct size studies (Table 1).

For delayed infarct size experiments, a significant difference in mean arterial pressure was evident between the DMSO vehicle group and the experimental group when wortmannin was administered 10 min before ischemia. No other significant differences were seen in hemodynamics for the delayed infarct size studies (Table 2).

Infarct size expressed as a percentage of the area at risk in rats (n = 6/group) treated with ethanol (EtOH) or DMSO vehicle or FIT (10 μg/kg) in the presence or absence of the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. Wortmannin was administered 90 min before FIT administration during the trigger phase [W(T)+FIT] or 10 min before ischemia during the end-effector phase [W(E)+FIT] or administered alone [W(T)] or 96 h later [W(E)]. Values are means ± SE. +P < 0.001 vs. vehicle. †P < 0.001, wortmannin administered with FIT as a trigger vs. wortmannin administered with FIT as an end-effector.

DISCUSSION

The novel finding of this study is that a single dose of the irreversible δ-opioid agonist FIT produces a delayed window of cardioprotection that persists 48 h longer than any known cardioprotective agent previously studied, including IP. The protective effect of FIT spans a range of administration from 10 s after reperfusion to 120 h before ischemia. This mechanism of delayed cardioprotection involves the PI3K pathway, which is an essential trigger and partial mediator and/or end effector of delayed opioid-induced cardioprotection.

Recently, the ability to protect the myocardium after reperfusion with brief, repetitive cycles of ischemia and reperfusion, coined postconditioning, has been shown to occur in dogs and rats (23, 44). Pharmacological agents also have shown the ability to reduce infarct size when administered at reperfusion, including adenosine and insulin (18, 43). Our data suggest that the efficacy of the selective δ-opioid agonist FIT to postcondition the myocardium reduces infarct size to an extent similar to that observed when FIT was administered either before ischemia or immediately before reperfusion. Interestingly, in a different study, morphine administered at 10 s after reperfusion failed to postcondition the myocardium (14). The differences in the ability of morphine and FIT to postcondition the myocardium may be caused by the greater selectivity and preference of FIT to bind to the δ-opioid receptor compared with morphine. Morphine may have less selectivity for the δ-opioid receptor (16).

The ability of FIT to reduce infarct size up to 120 h after administration makes it the first pharmacological agent known to extend the window of cardioprotection >72 h. Interestingly, adenosine is the only pharmacological agent previously shown to cause cardioprotection up to 72 h as assessed by infarct size (3). Both the KATP channel opener diazoxide and δ-opioid agonist TAN-67 have a significantly shorter window of cardioprotection, lasting 24 and 48 h, respectively (10, 38). Human studies with tissue taken from the right atrial appendage of patients with triple vessel coronary artery disease have shown that both adenosine and phenylephrine can cause protection from simulated ischemia-reperfusion up to 72 h after administration (27). The selective δ-opioid agonists BW373U86, SNC-80, and TAN-67 all produced similar infarct size reduction in previous studies at 24 h, with the infarct size reduction comparable to FIT-induced infarct size reduction at 96 h (31, 32). Interestingly, in the present study, BW373U86 (10 μg/kg) also reduced infarct size 96 h after administration, suggesting that the extended window of cardioprotection is perhaps a common mechanism among certain selective δ-opioid agonists. Collectively, these data suggest that pharmacological agents have different windows of protection that perhaps could be based on receptor selectivity and the dose of pharmacological agent administered.

The ability of FIT (10 μg) to cause acute cardioprotection is more efficacious, on the basis of the dose administered, compared with other δ-selective opioid agonists tested, such as BW373U86 (1 mg/kg) or TAN-67 (30 mg/kg), or nonselective agonists, such as morphine (0.3 mg/kg) when delivered as a bolus injection (10, 15). As an infarct size-reducing agent, the lower dose of FIT is just as effective as morphine or SNC-121 but not as effective as TAN-67. The greater infarct size reduction ability of TAN-67 may be due to 1) the generous dose used of TAN-67 (30 mg/kg), perhaps causing nonspecific effects to enhance infarct size reduction, 2) the differences in rat species (Wistar vs. Sprague-Dawley), or 3) the timing of the doses administered (1 h vs. 10 min before ischemia) (10).

An additional reason why differences in efficacy and infarct size-reducing ability are present for FIT compared with other δ-opioid agonists and morphine may be explained by the binding properties to the δ-opioid receptor for FIT compared with those to the other opioid agonists. A derivative of FIT, SUPERFIT, previously was shown to target the first intracellular loop to the middle of the third transmembrane helix (45). This region is different than the area targeted by the other δ-opioid agonists, BW373U86, SNC-121, and TAN-67, which target the fifth and seventh transmembrane regions of the third extracellular loop of the δ-opioid receptor (29). This could explain why FIT, as a delayed cardioprotective agent, has a greater reduction of infarct size after 96 h compared with BW373U86.

The inability of FIT to be protective at the higher doses studied (100 and 300 μg/kg) is not surprising, because previous studies have shown that other δ-opioid agonists produce a similar dose-dependent profile of infarct size reduction vs. δ-opioid agonist dose (31). The reason why higher doses of opioids are not protective is unknown. However, after significant activation of opioid receptors, negative feedback systems, which include cholecystokinin and excitatory opioid receptors coupled through Gs, are activated (41). Hence, the ability for opioids to induce cardioprotection may be based on the careful balance between exogenous opioid administration and endogenous antiopioid systems.

The ability of FIT to produce the maximal effect after 96 h, which is only submaximal after 48 or 72 h, also may be based on the balance between exogenous opioid administration and endogenous antiopioid systems. More studies concerning the time-dependent differences in infarct size for this agent need to be conducted. However, the time-dependent response on infarct size is not unique, because both κ-selective opioids and KATP channel openers produce a similar outcome of infarct size over time (8, 36).

The extended window of protection provided by FIT provides a significant advantage over IP, for which attempts in all species have failed to shown an extension of protection >72 h (2). The delayed protection by FIT at 96 h also displayed an infarct size reduction greater than the acute protective effects of FIT. These findings also suggest that opioids induce delayed protection perhaps by a different mechanism than IP, because previous studies have shown that delayed protection by IP is significantly less than the acute IP-induced cardioprotective effect (2).

For opioid-induced delayed cardioprotection, previous characterization has shown that important components of the trigger phase include the sarcolemmal KATP channel, MEK1/2, and p38, with components of the end effector being the mitochondrial KATP channel, 12-lipoxygenase, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) (11, 25, 31–33, 37). Traditionally, IP-induced delayed cardioprotection has exhibited an iNOS-dependent mechanism as an end effector at 24 h (5). Interestingly, the same laboratory recently showed that 72 h after IP, neuronal NOS but not iNOS was identified as the end effector (42). These data indicate the importance of developing models with an extended delayed cardioprotective window to discern the components involved in the trigger, mediator, and end-effector phases of cardioprotection. The length of a FIT- or BW373U86-induced window of cardioprotection will be a valuable tool in discerning the mechanisms involved in opioid-induced delayed cardioprotection.

Mechanistically, we found that the trigger phase of FIT-induced delayed cardioprotection appears to be dependent on the PI3K pathway to initiate pathways that are downstream of PI3K to be cardioprotective. These results are similar to those found in a previous study of delayed IP, where IP-induced delayed cardioprotection at 24 h was found to be abolished by pretreatment with wortmannin (24). Another salient finding is that the PI3K pathway is also required as a partial end effector of opioid-induced delayed cardioprotection, because wortmannin partially abrogated FIT-induced cardioprotection when administered 96 h after FIT, just before index ischemia. The contribution of PI3K as an end effector for IP is also unknown.

Previously, phosphorylation of both Akt and GSK-β, downstream mediators of PI3K, have been shown to be important in cardioprotection (20, 30). Addition of a constitutively active adenoviral Akt to the myocardium can cause protection and infarct size reduction 24–48 h after delivery (28, 30). IGF-1 and IP also have been shown to cause cardioprotection by Akt phosphorylation; both cardioprotection and Akt phosphorylation were abrogated in the presence of wortmannin (12, 24). Akt may act as a trigger of cardioprotection via multiple protein interactions, including its ability to induce transcriptional and antiapoptotic proteins, which includes the phosphorylation of GSK-β (17).

Phosphorylation of GSK-β, which produces its inactivation, previously has been shown to be important in mediating both IP- and opioid-induced infarct size reduction, with this cardioprotection mimicked by selective GSK inhibitors (15, 40). Interestingly, GSK-β phosphorylation may be a valuable convergence point for multiple cellular pathways including PI3K, protein kinase C, and targets of rapamycin-dependent pathways activated by both pharmacological agents and IP (15, 20, 40). It is also evident in nonmycoardial cells that GSK-β inhibition subsequently results in the activation of many diverse cellular signaling pathways, including the putative substrates heat shock factor-1 and NF-κB (19). These two substrates are of interest because both lead to induction of proteins found to be end effectors of delayed cardioprotection, such as heat shock protein-70, COX-2, and iNOS (1).

One of the established end effectors in opioid-induced delayed cardioprotection is NF-κB-induced COX-2 (26, 34, 37). Interestingly, COX-2 was recently shown to be regulated by the PI3K pathway in a variety of nonmyocardial cell lines (6, 26, 35, 39). Inhibition of GSK-β by lithium has been shown to elevate COX-2 expression and COX-2 promoter activity, whereas conversely, expression of a dominant negative Akt mutant or wild-type GSK-β suppressed COX-2 promoter activity (39). COX-2 induction by either the selective ATP-sensitive GSK-β inhibitors or lithium also was found to be mediated by increased NF-κB activity (35). Therefore, a possible link between the trigger and the induction of the end-effector proteins may involve GSK-β phosphorylation, and this cause and effect needs to be studied in more detail.

Our study is not without potential limitations, including the recognition that this is the first in vivo study conducted for FIT. Therefore, the half-life of FIT is unknown, and it is feasible that the irreversible nature of FIT maintains the ability of this agent to cause the extended protection. Because of the irreversible nature of FIT, we also used an irreversible antagonist of the PI3K pathway, wortmannin. Hence, we are confident that for our mechanistic studies, PI3K was inhibited for as long as FIT was irreversibly bound to the δ-opioid receptor.

In conclusion, we have found that a novel δ-selective agonist, FIT, has a cardioprotective window that extends from reperfusion to 120 h after administration. The trigger and end-effector phases of FIT-induced cardioprotection are also PI3K pathway dependent.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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